The primary focus of this project has evolved over the past year to cover studies of DNA polymerases and the mechanisms by which they achieve a high degree of replication fidelity. The two major projects that we worked on during the past year are: 1. studies of the E. coli DNA polymerase III subunits epsilon and theta, and their interaction, and 2. structural NMR studies of the domains of human DNA polymerase lambda. The E. coli polymerase III system is the most extensively studied model system for a replicative polymerase, and the mechanisms by which it achieves high replication fidelity are of central importance in understanding the environmental causes of mutations. We have recently completed a structural analysis of epsilon186, the catalytic domain of the epsilon subunit. This determination involved a combination of molecular homology modeling, guided by NMR-derived NOE restraints and secondary structure predictions. More recently, we have studied the effects of theta on the NMR spectrum of isotopically labeled epsilon186 in order to characterize the binding region. A second project has involved the NMR characterization of DNA polymerase lambda, a recently identified human polymerase which is highly homologous with the repair enzyme, polymerase beta. We have recently determined the structure of the isolated 8 kD domain in solution, which contains the dRP lyase activity of the enzyme. As anticipated, the structure is very similar to the corresponding domain from pol beta.